The proposal is steroid enzymology research to establish a heretofore unrecognized pathway of 19-nor-delta 4-androgen biosynthesis and to isolate and characterize the non-aromatizing androgen 19-hydroxylase and 19-desmolase in mammalian adrenal glands. Specific aims are 1) identification of the new enzyme system in the dog, sheep cow and human adrenal glands, and in small experimental animals, 2) development of a rapid enzyme assay method, 3) elucidation of the molecular mechanism by which a high apparent kinetic isotope effect is operated in the steroid 19-hydroxylation, 4) solubilization, purification and reconstitution of the 19-hydroxylase/desmolase system, 5) formation of antibodies and immunochemical comparisons with aromatizing 19-desmolase (estrogen synthetase), and 6) elucidation of the molecular mechanism of non-aromatizing androgen 19-desmolation. 19-Nortesterone has potent biological activities. 19- Hydroxyandrostenedione is a hypertensinogen and secreted from the adrenal glands. It is also converted to estrogens. The properties and regulation of such synthetases may have important implications in the understanding of adrenal physiology and regulation of the reproductive process. A specific antibody is useful for morphological identification of steroidogenesis and has potential value for diagnostic use. Elucidation of the reaction mechanism is also significant to biochemistry and pharmacology. It will give new insights into our understanding of steroid enzymology, cytochrome P-450 catalyzed drug oxidations and carcinogensis and toward designs with the adrenal C21-steroid 19-hydroxylation leading to 19- nordesoxycorticosterone biosynthesis will also be carried out in order to evaluate the substrate specificity of the enzyme system. The methods involve synthesis of steroids with and without deuterium, tritium, carbon-14 and oxygen-18 labels at stereoselective and/or regiospecific positions, structural studies and isotope distribution analysis, incubations with various enzyme preparations, identification of metabolites, determination of kinetic isotope effects, solubilization, purification and reconstitution of the enzyme system, antibody production, and immunochemical characterization.